It is estimated that sugar chains are involved not only in life phenomena such as development, differentiation and cell recognition but also in occurrence and progress of inflammations, cancers, infections, auto-immune diseases and a number of other diseases [A. Kobata, S. Hakomori and K. Nagai: Glycobiology Series (1) to (6), Kodansha (1993), Glycobiology, 3, 97 (1993)].
Sugar chains exsist not only as glycoproteins, proteoglycans or glycolipids, in which they are added to proteins or lipids, but also as oligosaccharides.
The poly-N-acetyllactosamine sugar chain as the subject of the present invention is a sugar chain with the structure having N-acetyllactosamine as the repeating unit bound via β1,3 linkage [(Gal β1-4GlcNAc β1-3)n where n is 2 or more], and it exists not only in N-glycoside linked sugar chains and O-glycoside linked sugar chains on glycoproteins but also in sugar chains of glycolipids and in oligosaccharides.
The poly-N-acetyllactosamine sugar chain is synthesized by alternately reaction of β1,4-galactosyltransferases and β1,3-N-acetylglucosaminyltransferases. The gene coding for the former enzyme β1,4-galactosyltransferase has already been cloned, but the gene coding for the latter enzyme β1,3-N-acetylglucosaminyltransferase is still not cloned. With respect to β1,3-N-acetylglucosaminyltransferases having poly-N-acetyllactosamine synthesis-related activity, there are only reports on their partial purification resulting in no information of their amino acid sequences [J. Biol. Chem., 268, 27118 (1993), J. Biol. Chem., 267, 2994 (1992), J. Biol. Chem., 263, 12461 (1988), Jpn. J. Med. Sci. Biol., 42, 77 (1989)].
In some galactose residues in poly-N-acetyllactosamine sugar chains, an N-acetylglucosamine is bound via β31,6-linkage to synthesize poly-N-acetyllactosamine sugar chains having branched chains such as Galβ1-4GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAc. A glycosyltransferase transferring such branched β1,6-bound N-acetylglucosamine is β1,6-N-acetylglucosaminyltransferase (I-branching enzyme). A gene for this enzyme has also been cloned. The linear poly-N-acetyllactosamine sugar chain (i-antigen) is recognized by anti-i antibody and the branched poly-N-acetyllactosamine sugar chain (I-antigen) is recognized by anti-I antibody [J. Biol. Chem., 254, 3221 (1979)].
Saccharides such as fucose, sialic acid, N-acetylgalactosamine, galactose etc. or sulfate groups etc are attached to linear or branched poly-N-acetyllactosamine sugar chains, and a wide variety of cell-specific or period-specific sugar chains (e.g. functional sugar chains, blood type sugar chains, cancer-related sugar chains) are formed [A. Kobata, S. Hakomori, K. Nagai: Glycobiology Series (1) to (6), Kodansha (1993), Japan].
It is known that poly-N-acetyllactosamine sugar chains having a sialyl-Lewis x sugar chain structure at their termini are present on granulocytes, monocytes or activated T cells, and these sugar chains function as ligands for E-selectin or P-selectin as adhesive molecules and are involved in accumulation of leukocytes into inflammation sites [A. Kobata, S. Hakomori, K. Nagai: Glycobiology Series (1) to (6), Kodansha (1993), Japan].
It is known that Poly-N-acetyllactosamine sugar chains having a sialyl-Lewis x sugar chain structure at their termini are present on cancer cells such as colon cancer cells, and it is suggested that these sugar chains also function as ligands for E-selectin or P-selectin and are involved in metastasis of cancer cells [A. Kobata, S. Hakomori, K. Nagai: Glycobiology Series (1) to (6), Kodansha (1993)].
It is known that the structure of poly-N-acetyllactosamine sugar chain is changed in the process of embryonic development, cell differentiation or cell transformation [A. Kobata, S. Hakomori, K. Nagai: GlycobiologySeries (1) to (6), Kodansha (1993)]. While linear poly-N-acetyllactosamine sugar chains are expressed in human embryonic erythrocytes, branched poly-N-acetyllactosamine sugar chains are expressed in human adult erythrocytes [A. Kobata, S. Hakomori, K. Nagai: Glycobiology Series (1) “World of Various Sugar Chains” Kodansha (1993)]. ABO-type blood group antigens are expressed at the termini of poly-N-acetyllactosamine sugar chains in these erythrocytes. Expression of blood group antigens at each terminus of branched poly-N-acetyllactosamine sugar chain results in multivalent antigens to increase the ability of the antigens to bind to antibodies against blood group sugar chains more than 103-fold compared with that of the linear antigens.
It is known that a series of sugar-chain antigens are expressed in a regulated manner during developmental process of mouse early embryo. SSEA-1 (stage specific embryonic antigen-1) is a Lewis x sugar chain [Galβ1-4(Fuc α1-3)GlcNAc] which appears at the termini of poly-N-acetyllactosamine sugar chains, and expression of this antigen is initiated at the 8-cell stage, peaks at the morula stage, and disappears after the blastocyst stage [A. Kobata, S. Hakomori, K. Nagai: Glycobiology Series {circle around (3)} “Glycobiology of Cellular Society”, Kodansha (1993)]. The morula stage corresponds to the stage at which embryonic cells having increased merely numerically by repeated proliferation through cell division enter the blastocyst stage at which the cells come to have a differentiated “form”. Morula cells adhere to each other just before forming a blastocyst to cause cell compaction. If an oligosaccharide having SSEA-1 antigen is added, this cell compaction is inhibited and normal development thereafter is also inhibited [J. Exp. Med., 160, 1591 (1984)]. It is also known that adhesion of mouse teratocarcinoma cells is inhibited by anti-SSEA-1 antibody [A. Kobata, S. Hakomori, K. Nagai: Glycobiology Series {circle around (3)} “Glycobiology of Cellular Society”, Kodansha (1993)]. The foregoing indicates that the SSEA-1 antigen acts as an adhesive molecule or a sugar chain signal to play an important role in development of early embryos.
It is known that poly-N-acetyllactosamine sugar chains are expressed at higher levels in cancer cells than in their corresponding normal cells [J. Biol. Chem., 259, 10834 (1984), J. Biol. Chem., 261, 10772 (1986), J. Biol. Chem., 266, 1772 (1991), J. Biol. Chem., 267, 5700 (1992)]. It is known that if N-ras protooncogene is expressed in NIH3T3 cells, the molecular weight of N-linked sugar chain on cells is increased, and the cells attain invasive activity, and at the same time, the amount of poly-N-acetyllactosamine sugar chains in the N-linked sugar chains is increased and simultaneously β1,4-galactosyltransferase and β1,3-N-acetylglucosaminyltransferase activities involved in synthesizing poly-N-acetyllactosamine sugar chains are increased [J. Biol. Chem., 266, 21674 (1991)].
Galectins are a family of lectins with affinity for β-galactoside, and are involved in cell adhesion and signal transduction, and their relation with diseases such as cancers is also suggested [Trends in Glycoscience and Glycotechnology, 9, 9 (1997)]. Ten types of Galectins have been found in mammals. Out of them, galectin-1 and galectin-3 are known to bind with high affinity to linear poly-N-acetyllactosamine sugar chains, and specific glycoproteins containing these sugar chains are estimated to be ligands for these galectins [Trends in Glycoscience and Glyotechology, 9, 9 (1997), Trends in Glycoscience and Glycotechnology, 9, 47 (1997)].
Poly-N-acetyllactosamine sugar chains having sialic acid residues at their termini serve as receptors for mycoplasma and microorganisms [Acta Paediatrica, 82, 903 (1993)].
As described above, poly-N-acetyllactosamine sugar chains play important roles in forming core sugar chains of many functional sugar chains (e.g. selectin ligand sugar chains, receptor sugar chains for microorganisms and viruses, SSEA-1 sugar chains and cancer-related sugar chains) and blood group. sugar chains to present these sugar chains effectively.
Poly-N-acetyllactosamine sugar chains having sialyl Lewis x sugar chains are expected to serve as a pharmaceutical product having anti-inflammatory effect or inhibitory effect for cancer metastasis.
It is known that an Poly-N-acetyllactosamine sugar chain having multivalent (4) sialyl Lewis x oligosaccharides (tetrasaccharides) has activity as selectin antagonist even at a low concentration of 1/100 or less relative to a non-multivalent sialyl Lewis x oligosaccharide (tetrasaccharide) [J. Exp. Med., 182, 1133 (1995), Glycobiology, 6, 65 (1996), Glycobiology, 7, 453 (1997), Eur. J. Immunol., 27, 1360 (1997)]. Partially purified β1,3-N-acetylglucosaminyltransferase was used for synthesis of poly-N-acetyllactosamine sugar chains in these oligosaccharides, however supply of this enzyme is rate-limiting, making it difficult to synthesize a large amount of poly-N-acetyllactosamine sugar chains [Glycobiology, 7, 453 (1997)].
Alternatively, poly-N-acetyllactosamine sugar chains can also be chemically synthesized, but their synthesis requires very complicated steps [Tetrahedron Letter, 24, 5223 (1997)].
From the foregoing, a method of efficiently synthesizing poly-N-acetyllactosamine sugar chains has been desired.
It is known that in human milk there are various oligosaccharides having the structure of poly-N-acetyllactosamine sugar chain [Acta Paediatrica, 82, 903 (1993)]. It is considered that these oligosaccharides have the function of preventing infants from being infected with viruses or microorganisms as well as the function of neutralizing toxins. They also have the activity of promoting growth of Bifidobacteria as good enterobacteria. On the other hand, the types of oligosaccharides present in milk of animals such as cattle or mice are limited, and most of them are composed of lactose, and there are few oligosaccharides containing poly-N-acetyllactosamine sugar chains [Acta Paediatrica, 82, 903 (1993), J. Biol. Chem., 270, 29515 (1995)].
It would be considered significantly advantageous if we could produce efficiently various oligosaccharides that are contained in human milk and are comprising a poly-N-acetyllactosamine sugar chain, as well as milk containing them; however, such a method has not been known.
Poly-N-acetyllactosamine sugar chains are also important for stabilization of glycoproteins. Lysosome associated membrane glycoprotein-1 (lamp-1) and lysosome associated membrane glycoprotein-2 (lamp-2) are glycoproteins present in lysosomes (some are present even on cell surfaces), with which almost all the inner face of the lysosome membrane is covered. A lot of sugar chains (some of which contain poly-N-acetyllactosamine sugar chains) are attached to lamp-1 and lamp-2, thus preventing lamp-1 and lamp-2 from being decomposed by hydrolyzing enzymes in lysosomes. If human promyelocytic leukemia cell line HL-60 is treated with dimethyl sulfoxide, it differentiated into granulocyte, and in this differentiation process it is known that the number of poly-N-acetyllactosamine sugar chains added to lamp-1 and lamp-2 is increased, and simultaneously the metabolic rate (decomposition rate) of lamp-1 and lamp-2 is decreased [J. Biol. Chem., 265, 20476 (1990)].
Because poly-N-acetyllactosamine sugar chains contribute to protein stabilization, it is considered that a protein of interest can be stabilized by artificially adding poly-N-acetyllactosamine sugar chains to the protein. Further, the clearance rate of a blood protein from kidney is decreased with an increasing effective molecular weight of the protein, thereby it would be possible to lower the clearance rate of a protein of interest from kidney and to increase its stability in blood by artificially adding poly-N-acetyllactosamine sugar chains to the protein. Furthermore, by adding poly-N-acetyllactosamine sugar chains to a protein of interest, the protein could be targeted to specific cells.
It is reported that if F9 cells are treated with retinoic acid or Swiss 3T3 are treated with TGF-β, poly-N-acetyllactosamine sugar chains are added to sugar chains of membrane-bound glycoproteins in the cells [J. Biol. Chem., 268, 1242 (1993), Biochim. Biophys. Acta., 1221, 330 (1994)].
It is known that if N-ras protooncogene is expressed in NIH3T3 cells, the activity of β1,4-galactosyltransferase and β1,3-N-acetylglucosaminyltransferase having poly-N-acetyllactosamine sugar chains synthesis-related activity is increased and the amount of the poly-N-acetyllactosamine sugar chains in the N-linked sugar chain of membrane protein is increased [J. Biol. Chem., 266, 21674 (1991)].
If a core β1,6-N-acetylglucosaminyltransferase gene is expressed in T-cell line EL-4, the molecular weight of CD43, CD45 or CD44 as a membrane protein on cell surface is increased [J. Biol. Chem., 271, 18732 (1996)]. This would be because sugar chains synthesized by core 2β1,6-N-acetylglucosaminyltransferase serve as good substrates for β1,3-N-acetylglucosaminyltransferase involved in synthesizing poly-N-acetyllactosamine sugar chains.
It is known that the amount of poly-N-acetyllactosamine sugar chains added to lamp-1 or lamp-2 is increased when HL-60 cells are cultured at 27° C. [J. Biol. Chem., 266, 23185 (1991)].
However, it is not clear whether the above method is effective or not in host cells suitable for production of recombinant glycoproteins. Accordingly, for host cells (e.g. Namalwa cells, Namalwa KJM-1 cells, CHO cells etc.) suitable for production of recombinant glycoproteins, improvement of their ability to synthesize poly-N-acetyllactosamine sugar chains is an industrially important subject.
In consideration of the mechanism of the above-described inflammatory reaction and cancer metastasis, it is expected that the inflammatory reaction can be inhibited and the cancer metastasis can be prevented by inhibiting expression of poly-N-acetyllactosamine sugar chains. However, a method of efficiently inhibiting expression of poly-N-acetyllactosamine sugar chains is not known.
It is expected that inflammatory diseases or cancer malignancy can be diagnosed by examining expression of genes (e.g. a gene for β1,3-N-acetylglucosaminyltransferase as a key enzyme in synthesis of poly-N-acetyllactosamine sugar chains) involved in synthesizing poly-N-acetyllactosamine sugar chains or by examining expression of polypeptides coded by their genes in inflammatory leukocytes, cancer cells or in serum. However, such a method is not known.